Abstract

Non-claret disjunctional (Ncd) is a kinesin-related microtubule motor protein in Drosophila that functions in meiotic spindle assembly in oocytes and spindle pole maintenance in early embryos. The partial loss-of-function mutant ncd^D retains mitotic, but not meiotic, function. The predicted Ncd^D mutant protein contains a V556->F mutation in the putative microtubule binding region of the Ncd motor domain. Here we report an analysis of the properties of recombinant Ncd and Ncd^D proteins. A GST-Ncd^D fusion protein translocated microtubules asymptotically equal to 10-fold more slowly than the corresponding wild-type protein in gliding assays. The maximum microtubule-stimulated ATPase activity of an Ncd^D motor domain protein was reduced asymptotically equal to 3-fold and an asymptotically equal to 3-fold greater concentration of microtubules was required for half-maximal stimulation of ATPase activity, compared with the corresponding wild-type protein. The K~m for ATP and basal rate of ATP turnover were, in contrast, similar for the Ncd^D mutant and wild-type Ncd motor domain proteins. Pelleting assays demonstrated that the binding of the mutant Ncd^D motor protein to microtubules was reduced in the absence of nucleotide, relative to wild-type. The reduced velocity of Ncd^D translocation on microtubules is therefore correlated with reductions in microtubule-stimulated ATPase activity and affinity of the mutant motor for microtubules. The characteristics of the Ncd^D motor explain its meiotic loss of function, and are consistent with partial motor activity of Ncd being sufficient for its mitotic, but not its meiotic, role.